Vol. 133: 287-297,1996
MARINE ECOLOGY PROGRESS SERIES
Mar Ecol Prog Ser
1
Published March 28
Effects of high-molecular-weight dissolved organic
matter on nitrogen dynamics in the
Mississippi River plume
Wayne S. G a r d n e r l r * , Ronald Benner2,Rainer M. W. ~ m o n James
~ ,
B. Cotner, ~
Joann F. Cavalettol, Jeffrey R. Johnson4
r
'NOAA Great Lakes Environmental Research Laboratory, 2205 Commonwealth Blvd, Ann Arbor, Michigan 48105. USA
'Marine Science Institute, University of Texas at Austin, Port Aransas, Texas 78373, USA
3Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, Texas 77843, USA
4Cooperative Institute f o r Limnology and Ecosystem Research, University of Michigan, Ann Arbor, Michigan 48105, USA
ABSTRACT- The dynamics of N and ~ t interactions
s
with labile dlssol\ied organic C (DOC), bacteria,
and phytoplankton were studied to determine potential effects of dissolved organlc matter (DOM) a n d
light on N dynamics in surface waters of the Mississippi River (USA) plume in the Gulf of Mexico. Bacterial uptake of added labeled N compounds ( I N N . , + or "N-labeled dissolved free amino acids. DFAA)
was stimulated more by high-molecular-weight (HMW, > l kDa) DOM than by 101~-molecular-weight
(LMW. < l kDa) DOM. An ~ n d e xthat ~nverselyindicated the presence of labile DOC was defined as the
fraction of assimilated Amino acid-l5B thdt was Recovered as "N-.Ammonium (ANRA),follo~vlngthe
additions of high-levels (4 pM) of "N-DFAA. ANRA ratios were high In the absence of other available
carbon sources because heterotrophic bacteria were forced to use the added amino acids as a carbon
source for respiration rather than as a nutrient source for biomass formation. In dynamic light/dark
experiments, conducted with in situ populations of organisms, uptake rates of added 15NH4+were significantly enhanced both by the presence of light and by the addition of HMW DOM. Uptake rates of
added I5N-labeled DFAA were increased by the addition of HMW DOM but not by light. ANRA ratios
were consistently lower in the presence of added HMM1 DOM than in controls. Added HMW DOM thus
appeared to stimulate the incorporation of assimilated DFAA into bacterial biomass. Bacterial growth
rates were relatively high in both light and dark bottles with DFAA additions and in light bottles with
HMW DOM plus NH,' additions, but they remained comparatively low in dark bottles with added
NH,' These results are consistent with the idea that bacterial N dynamics in these euphotic waters may
be tightly coupled to photosynthetic activities over short time scales.
KEY WORDS: Dissolved organic carbon - Nitrogen cycling . Ammonium Bacteria - Phytoplankton
Arnlno acids
INTRODUCTION
A large fraction of organic material from primary
production in aquatic ecosystems is thought to cycle
through dissolved organic matter (DOM) and subsequently through heterotrophic bacteria (Scavia & Laird
1987, Chin-Leo & Benner 1992, Chrost & Rai 1993),but
biogeochemical mechanisms responsible for these
interactions in surface waters are not yet well defined
O Inter-Research 1996
Resale of full article not permitted
(Miinster & Chrost 1990). The bulk of the DOM in the
ocean is considered to be quite resistant to bacterial
breakdown (Menzel 1974 and references therein) but
a significant fraction of the DOM in surface waters can
be asssimilated or respired by bacteria within days
(Kirchman et al. 1991). Bacterial incubations with
isolated DOM that had been fractionated into highmolecular-weight (HMW, > l kDa) and low-molecularweight (LMW, c 1 kDa) components indicated that
HMW DOM supported much more bacterial growth
and respiration than LMW DOM (Amon & Renner
~
,
288
Mar Ecol Prog Ser 133: 287-297, 1996
these incubations by the absence of light and by the
1994) These results seem to argue against the pararemoval of organisms larger than bacteria. In the secdigm that bacterial growth in natural waters is primarond 'natural-biota' experiments, w e conducted light
ily supported by LMW DOM, but they could be biased
and dark 15N isotope addition experiments on unfilby the fact that fluxes of LMW DOM in the water are
tered surface waters in the presence and absence of
not necessarily reflected by their concentrations in the
added HMW DOM that had been isolated from seawater at the time of DOM isolation. If labile Lh4W
water at the site. These experiments included natural
DOM compounds are removed from the water as
organisms and allowed photosynthesis and associated
rapidly as they are produced, concentratlons could be
DOM production/microbial interactions to continue in
low In isolated fractions even though their fluxes are
quantitively important to bacterial/microbial foodweb
the lighted bottles.
dynamics under in situ conditions.
Comparison of results from these 2 types of experiChemical analysis of HMW DOM isolated from
ment provides insights about the dynamic role of
ocean water indicates that it has a high concentration
HMW DOM and light-driven photosynthetic processes
of carbohydrates and C:N ratios of about 15 (Benner et
in the short-term turnover of Inorganic and organic N
al. 1992). Similarly, HMW dissolved organic carbon
compounds. In this paper, w e specifically consider the
following questions for surface waters in the Missis(DOC) from Mississippi River (USA) plume surface
waters have C:N ratios ranging from 14 to 20 a s comsippi River plume: (1) is HMW DOM a source or sink
pared to ratios of 19 to 26 in the river (R. Benner
for dissolved inorganic N compounds? (2) Do bacteria
use different organic substrates for growth (biomass
unpubl. data). These ratios are much higher than ratios
for bacterial biomass, suggesting that the isolated
formation) and energy (metabolism) in surface waters?
(3) Are bacterial production and heterotrophic N
labile HMW DOM may be an important carbon source
cycling rates directly enhanced by photosynthetic profor bacteria (Sakugawa & Handa 1985, Pakulski & Benduction of available DOC or DON?
ner 1994) but less important as a N source. Bacteria
that grow on this material therefore must obtaln most
of thelr N from inorganic sources or from LMW organic
METHODS
N compounds that are not reta.ined by the ultrafiltration process (Amon & Benner 1994).
Study sites and DOC analysis. Experiments were
Recent studies with added 15N have demonstrated
that under natural light, significant dissolved organic
conducted on board the RV 'Longhorn' in July 1993 in
nitrogen (DON) is released from phytoplankton (Bronk
the northern Gulf of Mexico in the vicinity of the Mississippi River plume. Samples were collected at 2 sites
& Glibert 1993). This recently released DON may be
(Table 1) of intermediate salinity where surface-water
assimilated or metabolized by bacteria (Keil & Kirchman 1991, 1993, Simon & Rosenstock 1992) or possibly
primary production (Lohrenz et al. 1990), bacterioby phytoplankton (Palenik & More1 1990a, b) and replankton production (Chin-Leo & Benner 1992), and
incorporated into the food web via the microbial loop
nutrient cycling rates (Cotner & Gardner 1993) are
(Bronk et al. 1994). These apparent differences in the
high relative to corresponding rates in surrounding
dynamics of DOC and DON may be explained by a
waters. DOC was measured in treatment-bottle waters
partial chemical uncoupling of DON and DOC as
with a Shimadzu TOC 5000 analyzer (Benner & Strom
microbial substrates (Kirchman et al. 1991). For exam1993).
ple, rapid cycling of labile, photosynthetically produced LMW DOM, e.g
DFAA' may be an important cOmpOTable 1. Sample site locations and surface-water characteristics for the fractionnent of DON turnover (Fuhrman 19901
ated-DOM-bacterial (FDOMB)and natural-blota experiments Notc, the experimental prec~slonof the DOC measurements is less than 2 % coeffic~entof variation
Kirchman et al. 1990).
To gain further insights about N
transformations mediated by primary
FDOMB
Natural-biota
experiment
experiment
producers and heterotrophic bacteria,
we conducted 2 types of experiment:
July 23, 1993
Sampling date
July 14, 1993
in the first 'fractionated-DOM-bacterSampling coordinates
29' 10.3' N,
28" 49.5' N,
ial' experiments, w e examined micro91°29.8'W
92" 05.84' W
bial-nitrogen interactions with HMW
30
Temperature ( " C )
30
and LMW DOM, respectively, that had
24
Salinity (psu)
8
been isolated from the water at a disDOC conc.
LMW DOM: 175
No added H M W DOM: 246
crete tlme point. Autotrophic DOM
(pg-atom C I-')
I IMW DOM: 269
With added HMW DObI: 525
production was prevented during
Gardner et a1 . Effects of orga nic matter on nitrogen dynamics
Nitrogen transformation experiments. In the 'fractionated-DOM-bacterial' experiment, HMW ( > l kDa)
DOM was physically separated from LMW ( < l kDa)
DOM, diluted back to natural concentrations in artificial seawater, and incubated in the dark as previously
described (Amon & Benner 1994). Seawater was filtered through a 0.1 pm pore-size hollow-fiber filter to
remove particles, and the DOM was partitioned into
HMW and LMW fractions by tangential-flow ultrafiltration (Benner 1991). A 2.5 1 concentrate of HMW
DOM was then diluted to natural concentrations with
ca 15 1 of artificial seawater. LMW DOM remained in
the original seawater after ultrafiltration. Each fraction
was inoculated with a natural bacterial assemblage
(<0.6 pm) that had been isolated from the initial water
sample. The DOC concentrations in incubations with
HMW and LMW DOM were 269 and 175 pM C 1-',
respectively (Table 1 ) .
Isotope dilution and enrichment experiments, with
I5NH4+or ISN-labeled amino acid additions to the fractionated-DOM treatments, were conducted in the dark
at in situ temperatures. After natural bacteria had been
added to both treatments (Amon & Benner 1994), subsamples (70 ml) from each were placed in 75 m1 tissue
culture bottles and treated with 4 pM levels of either
15NH,+or I5N-labeled DFAA (MSD Isotopes MN-2625
Algal Amino Acid Mixture). Each isotope-addltlon
experimental treatment was run in triplicate bottles for
experimental replication. Ammonium and DFAA concentrations and ammonium isotope ratios ( [ ' s N ~ 4 ' ] :
[total NH,']) were monitored a few minutes after addition of the labeled con~poundsand at 2 or 3 additional
intervals over 20 to 24 h.
'Natural-biota' isotope dilution a n d enrichment
experiments were conducted similarly in a shipboard
incubator both under natural light and in the dark with
natural seawater (controls) a n d with natural seawater
treated with concentrated HMW DOM isolated from
the same sites (Table 1). An assumption for these
experiments is that NH,' can be assimilated by either
phytoplankton or bacteria, whereas DFAA are more
likely to be assimilated by bacteria (Wheeler & Kirchman 1986). Relative bacterial growth rates were measured after incubations of ca 24 h to provide insights
about the interactive effects of added HMW DOM, N
(in the form of NH,+ or DFAA), and light on bacterial
growth rates.
To measure dissolved NH,+ and DFAA concentrations and ['5NH,+]:[totalNH,+] ratios, 10 m1 of water
were sampled from each treatment and passed
through a 0.2 pm pore-size nylon filter (25 mm diameter). The first 3 m1 was used to rinse the filter a n d the
next 7 m1 was collected in a clean 8 m1 vial (Wheaton
# 224884). Ammonium and DFAA concentrations were
measured on-board ship (Gardner & St. John 1991),
289
and the remaining filtrate was frozen for later isotope
ratio analysis by high performance liquid chromatography (HPLC; Gardner et al. 1991, 1993). Community
ammonium regeneration and potential uptake rates in
the bottles were calculated from changes in dissolved
ammonium concentrations and isotope ratios over time
using the Blackburn-Caperon model (Blackburn 1979,
Caperon et al. 1979).
Calculation of the percentage of assimilated Amino
acid-''N Recovered a s l S N - A n ~ m o n i u n(ANRA)
~
in
DFAA addition experiments. Isotope enrichment
experiments with relatively high-level additions of
I5N-labeled DFAA can provide useful information on
the 'maximum uptake velocity' (V,,,,,) of DFAA by the
bacterial community (Wright & Hobbie 1965) a n d can
also provide a qualitative indication of the relative
availability of other naturally occurring labile DOC
substrates to the bacterial community (see discussion
below). These experiments differ from typical radioactive tracer studies of amino acid dynamics in that they
a r e used as a n indicator of the presence or supply rates
of labile organic carbon rather than as a tool to trace
the in situ dynamics of natural amino acids in the
water. The use of 'saturating' rather than 'tracer' concentrations of DFAA minimizes isotope dilution of the
added DFAA with natural DFAA and allows estimation
of V,,,,, for DFAA In the water. In addition, the fate of
I5N rather than of I4C or 3H from the added labeled
amino acids is followed. The concentrations of amino
acids (4 PM) that were added a r e high relative to natural concentrations of amino acids, but are reasonable,
relative to potential turnover rates, for these summer
experiments in Mississippi River plume surface waters
(Cotner & Gardner 1993).
The ANRA ratio provides a n index that can be
assumed to be inversely related to the amounts or supply rates of non-amino labile organic compunds that
are available to bacteria if effects of bacterial grazers
and uptake of ammonium by phytoplankton a r e
accounted for. If the added amino acids (C:N ratio =
4.30) become the predominant forms of carbon that are
available to bacteria, a high percentage of the amino
acid carbon will b e metabolized, to meet respiration/
deamination energy requirements, a n d a correspondingly high percentage of the I5N added as amino acids
will in turn be mineralized to ammonium ( e . g Zehr et
al. 1985. Goldman et al. 1987). For example, in the Mississippi River plume region, about 9 0 % of the 'assimilated' I5N that had been added as labeled amino acids
was recovered as 15NH4+in bottle experiments with
dark subsurface waters as compared to about 48% in
surface waters under natural light (Gardner et al.
1993). Similarly, organic substrate-depleted Lake
Michigan bacteria regenerated a n amount of ammonium approximately equivalent to the amount of added
Mar Ecol Prog Ser 133: 287-297, 1996
DFAA consumed (Gardner et al. 1987, 1989). Conversely, if other sources of labile carbon are available
to be metabolized, more of the amino-N can be incorporated into biomass (see 'Results').
ANRA, expressed as %, was calculated as follows:
ANRA = (([NHdt],xRI)- ([NH,+],xRi)]/'([AA]i-[AA10 x 100
where [NH,'], is the concentration of NH,' at the sampling point where DFAA had reached background (or
near-background) levels, R, is the isotope ratio of
['5NH,+]:[totalNH4+1at the same sampling point that
[NH,'], was determined, [NH4+],is the concentration of
NH4+at the initial sampling point, Ri = isotope ratio of
[15NH,+]:[totalNH,+] at the initial sampling point, [AAji
is the measured DFAA concentration at the initial sampling point, and [AAIfis the measured amino acid concentration at the sampling point where DFAA concentrations had reached near-background levels. In using
this equation, we assume that changes in DFAA levels
are caused by uptake of the added 15N-labeled amino
acids. If initial concentrations of DFAA are higher than
4 pM plus the background, i.e. if measurable natural
U b A A are present in the water in addition to the background fluorescent response typically observed for
non-labile compunds (Gardner & St. John 1991, Keil &
Kirchman 1993),the decreases can exceed 4 pM due to
the presence of non-labeled labile amino acids. When
measurable unlabeled DFAA were present in any of
the treatments, we assumed that [AA],- [AA], = 4 pM
for the calculation, since the change in 15N-labeled
amino acids cannot exceed the amount that was initially added. The background response was equivalent
to that from DFAA levels of 0.4 and 0.8 PM, respectively, in waters used for the fractionated-DOM-bacterial and natural-biota experiments. Note, ANRA ratios
should be considered as a qualitative rather than an
absolute indicator of substrate availability because
their values can also be affected to some extent by
other factors such as isotope dilution of the DFAA pool,
mineralization of assimilated amino acids by bacterial
grazers, and/or uptake of I5NH4+by phytoplankton in
experiments with natural assemblages of organisms.
To minimize the effects of these factors, we calculated
ANRA at the first point where DFAA were reduced to
near-background levels and conducted some expenments in the dark as well as under natural light conditions.
Bacterial abundances, growth rates, and amino acid
turnover rates. Bacterial abundances were estimated
with acridine orange staining and epifluorescence
microscopy of duplicate samples (Hobbie et al. 1977).
Relative bacterial production rates were estimated
from rates of incorporation of 3H-thymidine into DNA
(Fuhrman & Azam 1982). Samples were incubated in
duplicate with 20 nM (final concentration) of [methyl-
3 ~ ] t h y m i d i n e(TdR) (Amersham). Nucleic acid and
protein fractions were separated with a trichloroacetic
acid extraction procedure (Chin-Leo & Benner 1992).
Killed blanks were used to correct for abiotic adsorption to filters. In killed controls, formalin was added to
a final concentration of 4 % , 15 to 20 min prior to the
addition of radioisotope.
Amino acid turnover rates were estimated with a
mixture of 3H-labeled DFAA from algal protein
hydrolysate (Amersham) Tracer amounts (less than
1 nM final concentration) were added to duplicate
samples and i.ncubated for 10.0 min at ambient temperature. Incubations were termmated by filtration
onto a 0.2 pm pore-size filter (Millipore GSWP). Duplicate filter and filtrate samples were collected and
frozen prior to further analyses. Filters were dissolved
with 1 m1 ethylacetate before radioassay. Turnover
rates were corrected for respired amino acids by allowing a 1 m1 sample to equilibrate with 2 m1 of distilled
water through the gaseous phase by a modification of
the procedure of Ashcroft et al. (1972).The filtrate of a
labeled sample was preserved (2% formalin final concentration) and placed in a 1.5m1 open centrifuge tube
inside of a closed 20 m1 scintillation vial containing
distilled water. The respired tritium ('H-H20) was
allowed to equilibrate at 25°C for 1 mo. Samples from
the inner and outer vials were analyzed in a liquid
scintillation counter. All dpm (disintegrations per
minute) in the large vial were assumed to be from
3H-H20.The fraction of amino acids respired was calculated as (dpmDw)/(dpmp+ dpmp), where dpmDw,
dpmp, and dpmFare dpm in distilled water, on the filter
and in the filtrate, respectively. All samples were corrected for background levels of 3H-H,0 by analysis of
killed controls. Background levels were less than 5 %
of the total dpm. Incorporation was calculated as
dpmp/(dpmp+ dpmF),and efficiency was estimated as
Incorporation/(Incorpora.tion + Respiration).
RESULTS
Fractionated-DOM-bacterial experiment
Except for an increase at the second sampling point,
the concentrations of added NH,' remained relatively
constant in the seawater containing LMW DOM in the
15NH4+treatments (Fig. 1).However, in the water containing HMW DOM, NH,+was removed to near-background levels during the 20 h incubation. This result is
consistent with the hypothesis that HMW DOM is an
'available' organic substrate with a high C:N ratio that
causes microbes to assimilate NH,+ In contrast, LMW
DOM must ha.ve either been unavailable or had a relatlvely low C:N ratio so that the bacteria did not require
Gardner et al.. Effects of organic matter on nitrogen dynamics
HMW + 1 5 ~ H 4 +
LMW +"NH~+
9
1.0
'
.
0.8
--.
1
1
;
??
HMW + I 5 ~ - A A
LMW -'%V
06
AA
C
08
04
5
m
%
U
L-
z
0
5 10 15 20
0
5
\
00
10 15 20 25
Time (hours)
Time (hours)
Fig. 1. Time-course results for ammonium (e)and amino acid
(0)concentrations and '"NH,'isotope ratios (m) for the fractionated-DOM-bacterial e x p e r ~ m e n twith HMW and LMW
DOM treatment waters treated with either "NH.,'
or '"Nlabeled amino acids and incubated for 20 h
additional N. Removal patterns for I5N-labeled DFAA
were similar in both treatments. After an initial lag,
probably d u e to relatively low bacterial numbers,
removal rates were high; the added DFAA (4 PM) were
completely removed within about 20 h (Fig. 1). The
ANRA ratios were higher in the LMW DOM treatment
(40 %) than in the HMW DOM treatment (22 %) where
NH,+ concentrations did not appreciably increase
(Table 2 ) .
For the LMW DOM treatment, NH,' regeneration
rates decreased from about 60 nM h -' In the first 2 invervals to approximately 0 in the third interval (Fig. 2).
This trend may have reflected the depletion of labile
organic nitrogen. Ammonium regeneration rates for
the HMW DOM treatment varied among replicate
measurements and did not show significant changes
Table 2. Summary of ANRA values ? SE (n In parentheses)
expressed as a percentage for the different amino acid addltion treatments in the the fractionated-DOM-bacterial
(FDOMB) and natural-biota experiments See text for ANRA
calculation procedure
Dark
FDOMB
Fig. 2 Mean potential uptake and regeneration rates for the
ammonium-additlon experiments during incubations for
HMW DOM (0)and LMW DOM (e)treatments calculated
from data presented In Fig. 1. Rates were calculated from
changes in ammonlum concentrations a n d isotope ratios
using the model of Blackburn (1979)
between initial and final time points. Calculated uptake rates for NH,' ranged from < O during the first interval for both treatments, when bacterial abundances
were relatively low, to about 0.2 pM h-' for the second
interval and to 0.3 pM h-' for the HMW DOM at the
third interval (Fig. 2). Uptake rates decreased after 6 h
for the LMW DOM treatment but continued to increase
during the second interval in the HMW DOM treatment, reflecting changes in bacterial nutritional status
for the 2 types of treatment (Amon & Benner 1994).
Relative bacterial growth rates a n d amino acid incorporation a n d respiration rates, determined with tracer
levels of %I-labeled DFAA (Cotner & Gardner 1993),
were measured after inc.ubating the fractionated material for 1 d . Bacterial abundances a n d thymidme incorporation rates were both substantially higher in the
HMW DOM treatments than in the LMW DOM treatments (Fig. 3a]. Amino acid incorporation and respiration rates, and amlno acid incorporation efficiencies,
were all higher in the HMW DOM treatments than in
the LMW DOM treatments (Fig. 3b).
Light
HMW
LMM!
22 i 3 (3) 40 + 2 (3)
flklW
Control
Control
.HMW
Natural-biota 36 i 2 (2) 65 r 2 (3) 27 t 3 (3) 61 + 4 (2)
Natural-biota experiment
Ammonium was removed fastest from the lighted
bottles with added HMW DOM a n d slowest from control water in the dark (Fig. 4). More NI-I,' was removed
Mar Ecol Prog Ser 133: 287-297, 1996
DARK
8
LMW
0.6
r;
c
c
.-0
El
NATURAL LIGHT
CONT. +NH;
CONT. +NH,+
1 .o
0.8
0.6
0.4
0.2
0.0
HMW
B
HMW
0.5
4
CONT.+AA
0.4
U
0
F
0.3
6.2
0.1
0.0
Amino acid
Respiration
Amino acld
Incorporation
Efficiency
Fig 3 (A) Bacterial abundances and thymidine incorporation
rates and (B) amino acid respiration and incorporation rates
and calculated incorporation effic~encies [ = Incorporation/
(Incorporation + Respiration)] for ?H-labeled amino acids after
treatment waters from the fractionated-DOM-bacterial experiment were incubated for about 24 h in the dark
from solution in bottles containing HMW DOM and in
lighted bottles than in control seawater treatments.
The same trends were seen in isotope ratios indicating
isotope dllution of 15NH,+ with I4NH,,+in all of the
treatments.
Ammonium regeneration and potential uptake rates
were calculated over the first 10 h because changes
over the first 2.5 h interval were not sufficient to produce a n adequate signal-to-noise ratio to allow recognition of significant trends. A 2-way ANOVA, testing
the effects of light ( p = 0.13) and HMW DOM additions (p = 0.22) over the 10 h interval, did not show
significant differences in NH,' regeneration rates.
The HMW dark treatment showed lower regeneration
"
0
5
10 15 2 0
0
5
10 15 2 0 25
Time (hours)
Fig. 4. Time-course results for ammonium ( 8 )and amino acid
( 0 )concentrations and 15NH,+ isotope ratios (m) for the natural-biota experiment conducted in the dark and under natural light and in the presence and absence of added HMW
DOM. Treatment waters were fortified with either ISNH4+or
"N-labeled amino acids and incubated for 23 h
rates than the light treatments but SEs for the other
treatments overlapped (Table 3). In contrast, uptake
rates during the same interval showed significant differences for both light (p = 0.0013) and HMW DOM
additions (p = 0.019) with progressive increases from
the dark control to the light HMW addition treatment
(Table 3). The presence of natural light had a slightly
greater effect than the addition of HMW DOM but
results from the 2 treatments were generally additive,
i.e. interactions between the 2 treatments
were
significant (p = 0.59). InterestTable 3. Ammonium regeneration and uptake rates (PM NH,' h-') i SE in
natural-biota experiments for Control and HMW treatment bottles incuingly, in the dark control treatments,
bated in the dark and under natural light conditions. Note, these data were
uptake and regeneration rates were not
calculated from the ammonium-addition expenments presented in Fig. 4
Significantly different from each other,
but in all other treatments potential
Dark
L~ght
uptake rates were significantly higher
Control
Control
HMW
HMW
than the regeneration rates (Table 3).
Removal rates for added DFAA, which
Regeneration rate 0.08 * 0.04 0.05 t 0.002
0.13 i 0.02 0.09 i 0.03
the
Of the
'OrnuUptake rate
nity for DFAA, were high, particularly in
Gardner et al.: Effects of organic matter on nitrogen dynamics
the HMW DOM treatments where DFAA were completely removed in about 10 h (Fig. 4). By comparison,
more than 20 h was needed for complete removal of
the added DFAA in the controls. The fact that HMW
DOM addition immediately enhanced the DFAA
removal rates (i.e. during the first interval) suggests
that the native bacterial populations were already
adapted to using DFAA at relatively high rates in the
presence of natural HMW DOM. The DFAA removal
patterns tvere similar in light and dark bottles for both
control and HMW DOM treatments.
Isotope ratios of NH,' generally increased in proportion to decreases in lSN-labeled DFAA concentrations. However, NH,+ accumulation from DFAA
degradation was minimal in the treatments with
added HMW DOM. More NH,' accumulated in the
dark than in the light and in the controls than in the
waters treated with HMW DOM (Fig. 4). The ANRA
ratios were about 36 and 2 6 % in the dark and light
HMW DOM treatments a s compared to 64 and 61 %
in the corresponding control treatments (Table 2). In
the treatments with added HMW DOM, the isotope
ratios increased proportionately with the decreases in
DFAA concentrations, but then decreased slightly
after the DFAA were depleted to background levels
(Fig 4 ) . This decrease can be attributed to isotope
dilution of the '"H4+ that was in solution after degradation of the ISN-labeled DFAA. Final NH,' concentrations were similar in comparable treatments
whether NH4+or DFAA was the initial source of available N (Fig. 4).
Effects of light and HMW DOM additions o n relative
bacterial growth rates
293
DISCUSSION
Is HMW DOM a source o r sink for dissolved
inorganic nitrogen compounds?
In agreement with expectations from the chemical
analyses showing a high C:N ratio and carbohydrate
content for HMW DOM (Benner et a1 1992, Pakulski &
Benner 1994), our data suggest that HMW DOM is a
source of C for heterotrophic bacteria that in turn form
a biological sink for NH4+.Bacterial abundances and
growth rates increased much more in the presence of
isolated HMW DOM than in the presence of natural
levels of LMW DOM (Amon & Benner 1994). In all of
our experiments with HMW DOM, NH,' concentrations decreased more in the presence of added HMW
DOM than they did in its absence. The contention that
HMW DOM is an available C source for bacteria is
supported by the fact that it caused increased uptake
of both forms of N in the natural-biota experiments but
did not enhance NH4+regeneration rates.
The presence of HMW DOM in experiments with
DFAA additions greatly enhanced the V,,,,, of the bacterial community for DFAA uptake and consistently
caused ANRA ratios to be lower than was observed in
comparable control bottles without HMW DOM additions. In the fractionated-DOM-bacterial experiment,
removal patterns for ISN-labeled DFAA were similar in
both treatments but ANRA ratios were higher with
LMW DOM (40%) than with HMW DOM ( 2 2 % ) . The
HMW DOM apparently enabled more of the DFAA-N
to be incorporated into biomass than did the LMW
DOM. Thymidine incorporation rates and DFAA incorporation and respiration rates tvere all higher in the
HMW DOM treatments than in the LMW DOM treatment. The incorporation efficiency (ratio of DFAA
incorporation rate: DFAA incorporation rate + respiration rate) was also slightly higher for the HMW DOM
than for the isolated LMW DOM. These results are
consistent with the idea that HMW DOM is a bioavailable substrate with a relatively high C : N ratio (Amon &
Benner 1994) but did not provide evidence for high
reactivity of isolated LMW DOM.
Relative bacterial growth rates were measured in all
of the above treatments that had been enriched with
15NH,+ or I5N-labeled DFAA and incubated for about
24 h under light (natural) or dark conditions (Table 4 ) .
The highest growth rates were observed in bottles
incubated in the light with added HMW DOM and
DFAA. Similar high growth rates were observed in
lighted bottles containing HMW DOM
and added NH,' Much lower rates were
observed in the dark bottles with NH.,*
Table 4 . Relative bacterial growth rates (PM TdR h-') + SE in control and
HMW natural-biota experiments incubated in the dark and under natural
additions (with or without added HMW
light conditions with 4 pA4 additions of ' 5 ~ ~ or
4 +"N-labeled amino acids.
DOM, and in the light treatment without
See Fig. 4 legend for information on sample treatments
HMW DOM. In contrast, relatively high
arowth rates. c o m ~ a r a b l eto results for
I
Dark
Llght
light bottles, were observed in dark botControl
HMW
Control
HMW
tles with added DFAA. Incubations in the
light with added NH,' and HMW DOM
33i8
26*9
4 7 t 12
131+32
produced approximately the same result
+ 15N-labeled amino acids 106 i 10 125 * 1 4 92 + 9
156 * 2
as adding the DFAA directly.
d
294
Mar Ecol Prog Ser 133: 287-297, 1996
In our natural-b~otaexperiment, ANRA in the dark
was higher in the control treatments (64 % ) than in the
ones treated with HMW DOM (36%). Both of these
percentages were higher than the corresponding conversions observed in the fractionated-DOM-bacterial
expenm.ent. This difference could result from sampling site differences or more likely from the fact
that remineralizing micrograzers were present In the
natural-biota experiment but were removed before
incubations in the fractionated-DOM-bacterial experiment. Likewise, in the light ANRA was higher in the
control ( 6 1 % ) than in the HMW DOM treatment
(26%). The observation that ANRA was consistently
higher in control treatments than in the HMW DOM
ones suggests that HMW DOM enhanced incorporation of amino acid nitrogen into bacterial biomass (vs
respiratory loss of the organic nltrogen as NU,'). The
similarity of ANRA values in comparable light and
dark treatments (Table 2) indicates that a relatively
small portion of the mineralized ammonium was
taken up by phytoplankton before ANRA measurements were made.
Do bacteria use different organic substrates for
growth and energy in surface waters?
The experiments described here in conjunction with.
the corresponding data of Amon & Benner (1994) suggest that the dynamics of DOC and DON are partially
uncoupled (Kirchman et al. 1991). The HMW DOM
that can be isolated by ultrafiltration consists largely of
carbohydrates and has a relatively high C:N ratio (Benner et al. 1992). This fresh material appears to be an
important source of C for bacteria but does not appear
to provide sufficient N for bacterial growth. Thus. N
must come from inorganic forms or from other sources
such as DFAA or peptides (Coffin 1989, Keil & Kirchman 1991, Rosenstock & Simon 1993). Of course, the
dynamics of DOC and DON cannot be completely
uncoupled if labile LMW DON compounds such as
DFAA or peptides are quantitatively important
metabolites because these compounds contain both
organic C and N.
Studies examining the fate of I5NH4+and '"0,- in
isotope addition experiments suggest that a substantial
fraction of the 15Nincorporated into organisms under
natural light is released as "N-DON with relatively
short turnover times (Bronk & Glibert 1993, Bronk et
al. 1994). This DON release can result as a by-product
of feeding by zooplankton or protozoans (Nagata &
Kirchman 1991, Bronk & Glibert 1993) that tend to
release HMW DON, or from passive release of LMW
DON by autotrophs (Hellebust 1974, Bronk & Glibert
1993). Our results in combination with previous analy-
sis of HMW DOM (e.g.Benner et al. 1992) suggest that
any labile DON that is produced must occur mainly as
short-lived LMW DOM. If substantial DON is released
as proteins that are stable for more than a few hours,
one would expect HMW DOM isolated by ultrafiltration to have a lower C:N ratio than has been observed
(Benner et al. 1992).Likewise, HMW DOM would tend
to be a source rather than a sink for nitrogen in dark
bottle incubations (Amon & Benner 1994).
Are bacterial production rates and heterotrophic N
cycling rates directly enhanced by photosynthetic
production of available DOC or DON?
Bacterial numbers and growth rates (Amon & Benner
1994) and NH,' uptake rates were directly enhanced
by the presence of fresh HMW DOM. The rapid
removal of DFAA in both the HMW and L M W treatments in the fractionated-DOM-bacterial experiment
suggests that the V,,,, for DFAA uptake was high, as
would be expected at the experimental temperature of
3 F 2 , As mentioned above, oniy d b v ~ 2~2i % u i iile
assimilated ''N from the DFAA was recovered as
"NH,' in the HMW DOM treatment as compared to
40% for the LMW DOM treatment. The presence of
HMW DOM as a C source apparently allowed the bacteria to use the DFAA for biomass incorporation more
efficiently than when added DFAA alone were the
main source of carbon.
The fractionated-DOM-bacterial experiment did not
show greatly enhanced microbiological activity or N
cycling rates in the presence of isolated LMW DOM.
This observation would seem to argue against the paradigm that microbial processes are driven by LMW
organic compounds. However, another feasible explanation for this observation is that bacterial activity
decreases when bacterial processes are experimentally uncoupled from autotrophic processes that serve
as short-term sources of labile organic materials to the
bacteria. Recent studies have shown a close relationship between bacterial production and photosynthetic
processes (Cole et al. 1982, 1988, Coffin et al. 1994).If
turnover of the la.blle organic compounds is rapid, and
if bacterial uptake rates are as rapid as production
rates of the labile compounds, steady-state concentrations of some materials (e.g. DFAA) remain very low
even though the actual fluxes of the materials are high.
Our natural-biota experiments allowed comparison of
N cycling rates during times when active autotrophic
production of bacterial substrates should have
occurred (light experiments with natural biota) to those
when new production of labile organic substrates
should have been low (fractionated-DOM-bacterial
and dark natural-biota experiments)
Gardner et d..
Effects of organlc, matter on nitrogen dynamlcs
Our ANOVA results indicate that the presence of
both light and added HMW DOM increased NH,'
uptake and isotope d~lutionrates. Light had a slightly
greater effect on NH,' uptake rates than added HMW
DOM but the effects were comparable and non-interactive. These results suggest that both bacteria and
phytoplankton were actively assimilating NH,' in the
l ~ g h with
t
added HLIW DOM.
The minimal d~fferences between light and dark
DFAA removal patterns that were observed both In
the presence and absence of added HMW DOM indicate that heterotrophic bacter~a rather than phytoplankton were primarily responsible for DFAA
uptake. The high removal rates (I.e. V,,,,,) for the
added DFAA, part~cularlyIn the presence of added
HMW DOM, suggest that the bacteria may have been
accustomed to turning over relatively large quantities
of DFAA under natural conditions even though DFAA
concentrations are normally low. The HMW DOM
apparently increased the efficiency of DFAA uptake
by heterotrophic bacteria as DFAA uptake rates
approximately doubled when isolated HMW DOM
was added. This increase in DFAA uptake rates was
already observed during the first 4 h incubation interval, an indication that the bacteria were likely already
adapted to using t h ~ scombination of substrates (1.e
DFAA with HMW DOM). The ANRA values were
consistently lower in HMW DOM treatments than in
controls, suggesting that the I-IMW DOM stimulated
the incorporation of amino acid-N into bacterial biomass. A conceptual model that would explain these
observations is that DFAA, produced by autotrophic
processes, are rapidly used by heterotrophic bacteria
mainly for biomass formation, particularly when labile
HMW DOM with a high C:N ratio is available as a
source of energy for respiration.
Examination of bacterial growth rates in the different 15N-addition treatments provides revealing insight
about the effects of DFAA and autotrophic production
processes on bacterial production in these waters.
Addition of DFAA stimulated growth rates in all treatments as may be expected if DFAA are a preferred
source of N by bacteria (e.g.Kirchman et al. 1989, Keil
& Kirchman 1991) The addition of HMW DOM
appeared to stimulate bacter~algrowth rates In the
presence of added DFAA in the l ~ g h and
t
dark and in
the presence of NH,' in the light but not in the dark.
Lowest bacterial growth rates were observed for NF3,'
addit~onsIn the dark and only sl~ghtlyhigher growth
rates were observed for NH,' add~tionsin the light
treatment without added HMW DOM. We unfortunately did not measure bacterial production rates in
comparable bottles without N additions and therefore
could not determine the extent that NH,' addition
stimulated bacterial production rates over those with-
295
out nitrogen additions. However, our results show that
NH,' additions to HMW DOM in the light can clearly
stimulate bacterial production more than the same
additions in the dark.
The increased bacterial production rates in the
lighted HMW DOM ti-eatment suggest that autotrophic
processes interacted with HMW DOM to enhance bact e r ~ a lgrowth rates. Possibly, there was autotrophic
conversion of the 15NH,' to readily available 01-ganicN
compounds such as DFAA in the presence of light and
a source of labile carbon. Alternatively, the phytoplankton may have provided the bacteria with other
'growth factors' that allowed them to efficiently assimilate NH in the presence of HMW DOM. These data
are interesting relative to die1 d~fferencesin 'bacter~al
growth capacity' observed for bacteria in microcosn~s
(Zweifel et al. 1993). The growth capacity, defined as
the growth yield of bacteria in filtered water relative to
natural bacterial abundances in the same water,
tended to be lower at night than during the day
(Zwelfel et al. 1993). Also, In nuti-lent-enriched mesocosms, bacterial production, O2 consumption, and
growth eff~ciencieswere highest during daylight or
early evening when substrate availability was highest
(Coffin et a1 1994). In Chesapeake Bay, amino acid
uptake rates were also higher in daylight than at n ~ g h t
(Glibert et al. 1991).
The above experiments suggest that nutrlent
cycllng processes in Mississippi River plume surface
waters are closely linked to light-driven organic matter production or conversion by the photosynthetic
process. The ANRA index appears to effectively indicate the relative degree that labile DOC was available
to support bacterial respiration in the different treatments. HMW DOM appears to be a more important
source of C and energy for bacteria than LMW DOM.
In contrast, LMW DOM may be an important N source
for bacteria, as suggested by high V,,,, values for
DFAA a n d increased incorporation rates of high levels
of DFAA into biomass in the presence of HMW DOM.
Our data agree with the hypothesis that HMW DOC
con~poundsprovide a carbon source to 'fuel' bacterial
growth but that rapidly-recycling LMW organic N
compounds ( e . g . DFAA), or possibly some other
growth factors plus NH,', are needed for bacterial
biomass formation in surface waters of the Mississippi
River plume.
,+
Acknowledgcnients This research was sponbored by the
KOAA Coastal Ocean P ~ o g r a m through the Nutrlent
Enhanced Coastal Ocean Plogram (NECOP) We thank the
crew of the RV 'Longholn' for s h ~ psupport, Lynn Herche for
s t a t ~ s t ~ canalyses,
al
and Harvey Bootsina, Peter Landrum, and
David K ~ ~ c h ~ nfor
a nconstructive comments on the manuscript Thls paper is CLERL Contribution No 937
Mar Ecol Prog Ser 133: 287-297, 1996
296
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This ~lrticlewas presented by B. & E Sherr (Senior Editorial
Advisors), Corvallis, Oregon, U S A
Manuscr~ptfirst received: June 12, 1995
Revised version accepted: October 4 , 1995